Dennis E Bulman

Brachydactyly type A1 is an autosomal dominant disorder primarily characterized by hypoplasia/aplasia of the middle phalanges of digits 2–5. Human and mouse genetic perturbations in the BMP-SMAD signaling pathway have been associated with many brachymesophalangies, including BDA1, as causative mutations in IHH and GDF5 have been previously identified. GDF5 interacts directly as the preferred ligand for the BMP type-1 receptor BMPR1B and is important for both chondrogenesis and digit formation. We report pathogenic variants in BMPR1B that are associated with complex BDA1. A c.975A>C (p.(Lys325Asn)) was identified in the first patient displaying absent middle phalanges and shortened distal phalanges of the toes in addition to the significant shortening of middle phalanges in digits 2, 3 and 5 of the hands. The second patient displayed a combination of brachydactyly and arachnodactyly. The sequencing of BMPR1B in this individual revealed a novel c.447-1G>A at a canonical acceptor splice site of exon 8, which is predicted to create a novel acceptor site, thus leading to a translational reading frameshift. Both mutations are most likely to act in a dominant-negative manner, similar to the effects observed in BMPR1B mutations that cause BDA2. These findings demonstrate that BMPR1B is another gene involved with the pathogenesis of BDA1 and illustrates the continuum of phenotypes between BDA1 and BDA2.

Glycyl-tRNA synthetase (GARS) is an aminoacyl-tRNA synthetase (ARS) that links the amino acid glycine to its corresponding tRNA prior to protein translation and is one of three bifunctional ARS that are active within both the cytoplasm and mitochondria. Dominant mutations in GARS cause rare forms of Charcot-Marie-Tooth disease and distal spinal muscular atrophy.

Background: There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. Results: A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. Conclusions: The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups.

Despite remarkable advances in genetic testing, many adults with syndromic epilepsy remain without a molecular diagnosis. The challenge in providing genetic testing for this patient population lies in the extensive genetic heterogeneity associated with epilepsy. Even for the subset of epilepsy patients that present with a defining feature, such as microcephaly, the number of possible genes that would require interrogation by Sanger sequencing is extensive and often prohibitively expensive.

Background: To elucidate the genetic basis of a novel neurodegenerative disorder in an Old Order Amish pedigree by combining homozygosity mapping with exome sequencing. Methods and results: We identified four individuals with an autosomal recessive condition affecting the central nervous system (CNS). Neuroimaging studies identified progressive global CNS tissue loss presenting early in life, associated with microcephaly, seizures, and psychomotor retardation; based on this, we named the condition Autosomal Recessive Cerebral Atrophy (ARCA). Using two unbiased genetic approaches, homozygosity mapping and exome sequencing, we narrowed the candidate region to chromosome 11q and identified the c.995C > T (p.Thr332Met) mutation in the TMPRSS4 gene. Sanger sequencing of additional relatives confirmed that the c.995C > T genotype segregates with the ARCA phenotype. Residue Thr332 is conserved across species and among various ethnic groups. The mutation is predicted to be deleterious, most likely due to a protein structure alteration as demonstrated with protein modelling. Conclusions: This novel disease is the first to demonstrate a neurological role for a transmembrane serine proteases family member. This study demonstrates a proof-of-concept whereby combining exome sequencing with homozygosity mapping can find the genetic cause of a rare disease and acquire better understanding of a poorly described protein in human development.

Background: We recently described a novel autosomal recessive neurodevelopmental disorder with intellectual disability in four patients from two related Hutterite families. Identity-by-descent mapping localized the gene to a 5.1 Mb region at chromosome 16p13.3 containing more than 170 known or predicted genes. The objective of this study was to identify the causative gene for this rare disorder.Methods and results: Candidate gene sequencing followed by exome sequencing identified a homozygous missense mutation p.Gly46Arg, inTHOC6. No other potentially causative coding variants were present within the critical region on chromosome 16. THOC6 is a member of the THO/TREX complex which is involved in coordinating mRNA processing with mRNA export from the nucleus.In situhybridization showed thatthoc6is highly expressed in the midbrain and eyes. Cellular localization studies demonstrated that wild-type THOC6 is present within the nucleus as is the case for other THO complex proteins. However, mutant THOC6 was predominantly localized to the cytoplasm, suggesting that the mutant protein is unable to carry out its normal function. siRNA knockdown ofTHOC6revealed increased apoptosis in cultured cells.Conclusion: Our findings associate a missense mutation inTHOC6with intellectual disability, suggesting the THO/TREX complex plays an important role in neurodevelopment.

Methylmalonate semialdehyde dehydrogenase (MMSDH) deficiency is a rare autosomal recessive disorder with varied metabolite abnormalities, including accumulation of 3-hydroxyisobutyric, 3-hydroxypropionic, 3-aminoisobutyric and methylmalonic acids, as well as β-alanine. Existing reports describe a highly variable clinical and biochemical phenotype, which can make diagnosis a challenge. To date, only three reported cases have been confirmed at the molecular level, through identification of homozygous mutations in ALDH6A1, the gene encoding MMSDH. Confirmation by enzyme assay has until now not been possible, due to the extreme instability of the enzyme substrate.

Floating-Harbor syndrome (FHS) is a rare condition characterized by short stature, delays in expressive language, and a distinctive facial appearance. Recently, heterozygous truncating mutations in SRCAP were determined to be disease-causing. With the availability of a DNA based confirmatory test, we set forth to define the clinical features of this syndrome. Clinical information on fifty-two individuals with SRCAP mutations was collected using standardized questionnaires. Twenty-four males and twenty-eight females were studied with ages ranging from 2 to 52 years. The facial phenotype and expressive language impairments were defining features within the group. Height measurements were typically between minus two and minus four standard deviations, with occipitofrontal circumferences usually within the average range. Thirty-three of the subjects (63%) had at least one major anomaly requiring medical intervention. We did not observe any specific phenotype-genotype correlations. This large cohort of individuals with molecularly confirmed FHS has allowed us to better delineate the clinical features of this rare but classic genetic syndrome, thereby facilitating the development of management protocols.

Background: D-bifunctional protein (DBP) deficiency is typically apparent within the first month of life with most infants demonstrating hypotonia, psychomotor delay and seizures. Few children survive beyond two years of age. Among patients with prolonged survival all demonstrate severe gross motor delay, absent language development, and severe hearing and visual impairment. DBP contains three catalytically active domains; an N-terminal dehydrogenase, a central hydratase and a C-terminal sterol carrier protein-2-like domain. Three subtypes of the disease are identified based upon the domain affected; DBP type I results from a combined deficiency of dehydrogenase and hydratase activity; DBP type II from isolated hydratase deficiency and DBP type III from isolated dehydrogenase deficiency. Here we report two brothers (16½ and 14 years old) with DBP deficiency characterized by normal early childhood followed by sensorineural hearing loss, progressive cerebellar and sensory ataxia and subclinical retinitis pigmentosa. Methods and results: Biochemical analysis revealed normal levels of plasma VLCFA, phytanic acid and pristanic acid, and normal bile acids in urine; based on these results no diagnosis was made. Exome analysis was performed using the Agilent SureSelect 50Mb All Exon Kit and the Illumina HiSeq 2000 next-generation-sequencing (NGS) platform. Compound heterozygous mutations were identified by exome sequencing and confirmed by Sanger sequencing within the dehydrogenase domain (c.101C>T; p.Ala34Val) and hydratase domain (c.1547T>C; p.Ile516Thr) of the 17β-hydroxysteroid dehydrogenase type 4 gene (HSD17B4). These mutations have been previously reported in patients with severe-forms of DBP deficiency, however each mutation was reported in combination with another mutation affecting the same domain. Subsequent studies in fibroblasts revealed normal VLCFA levels, normal C26:0 but reduced pristanic acid beta-oxidation activity. Both DBP hydratase and dehydrogenase activity were markedly decreased but detectable. Conclusions: We propose that the DBP phenotype seen in this family represents a distinct and novel subtype of DBP deficiency, which we have termed type IV based on the presence of a missense mutation in each of the domains of DBP resulting in markedly reduced but detectable hydratase and dehydrogenase activity of DBP. Given that the biochemical testing in plasma was normal in these patients, this is likely an underdiagnosed form of DBP deficiency.